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We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele-dependent manner. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared with other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants. In addition, we nominate 17 variants (including 11 previously unreported) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies in primary and immortalized B cells. Thus, we uncover important insights into the mechanistic relationships among genotype, epigenetics, and gene expression in SLE and AI disease phenotypes.
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Infecciones por Virus de Epstein-Barr , Lupus Eritematoso Sistémico , Humanos , Sitios de Carácter Cuantitativo/genética , Histonas/genética , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , Lupus Eritematoso Sistémico/genéticaRESUMEN
Objective: To systematically characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate gene expression in an allele dependent manner. Methods: We designed a massively parallel reporter assay (MPRA) containing ~32K variants and transfected it into an Epstein-Barr virus transformed B cell line generated from an SLE case. Results: Our study expands our understanding of hQTLs, illustrating that epigenetic QTLs are more likely to contribute to functional mechanisms than eQTLs and other variant types, and a large proportion of hQTLs overlap transcription start sites (TSS) of noncoding RNAs. In addition, we nominate 17 variants (including 11 novel) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies primary and immortalized B cells. Conclusion: We uncover important insights into the mechanistic relationships between genotype, epigenetics, gene expression, and SLE and AI disease phenotypes.
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Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.
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OBJECTIVE: Sjögren's disease (SjD) is an autoimmune disease characterised by inflammatory destruction of exocrine glands. Patients with autoantibodies to Ro/SSA (SjDRo+) exhibit more severe disease. Long non-coding RNAs (lncRNAs) are a functionally diverse class of non-protein-coding RNAs whose role in autoimmune disease pathology has not been well characterised. METHODS: Whole blood RNA-sequencing (RNA-seq) was performed on SjD cases (n=23 Ro/SSA negative (SjDRo-); n=27 Ro/SSA positive (SjDRo+) and healthy controls (HCs; n=27). Bioinformatics and pathway analyses of differentially expressed (DE) transcripts (log2 fold change ≥2 or ≤0.5; padj<0.05) were used to predict lncRNA function. LINC01871 was characterised by RNA-seq analyses of HSB-2 cells with CRISPR-targeted LINC01871 deletion (LINC01871-/ -) and in vitro stimulation assays. RESULTS: Whole blood RNA-seq revealed autoantibody-specific transcription profiles and disproportionate downregulation of DE transcripts in SjD cases relative to HCs. Sixteen DE lncRNAs exhibited correlated expression with the interferon (IFN)-regulated gene, RSAD2, in SjDRo+ (r≥0.65 or ≤-0.6); four antisense lncRNAs exhibited IFN-regulated expression in immune cell lines. LINC01871 was upregulated in all SjD cases. RNA-seq and pathway analyses of LINC01871-/ - cells implicated roles in cytotoxic function, differentiation and IFNγ induction. LINC01871 was induced by IFNγ in a myeloid cell line and regulated by calcineurin/NFAT pathway and T cell receptor (TCR) signalling in primary human T cells. CONCLUSION: LINC01871 influences expression of many immune cell genes and growth factors, is IFNγ inducible, and regulated by calcineurin signalling and TCR ligand engagement. Altered LINC01871 expression may influence the dysregulated T cell inflammatory pathways implicated in SjD.
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Enfermedades Autoinmunes , ARN Largo no Codificante , Síndrome de Sjögren , Humanos , Interferones , ARN Largo no Codificante/genética , Calcineurina , Antivirales , Síndrome de Sjögren/genética , Autoanticuerpos , Inmunidad , Receptores de Antígenos de Linfocitos TRESUMEN
TNFAIP3/A20 is a prominent autoimmune disease risk locus that is correlated with hypomorphic TNFAIP3 expression and exhibits complex chromatin architecture with over 30 predicted enhancers. This study aimed to functionally characterize an enhancer â¼55 kb upstream of the TNFAIP3 promoter marked by the systemic lupus erythematosus (SLE) risk haplotype index SNP, rs10499197. Allele effects of rs10499197, rs58905141, and rs9494868 were tested by EMSA and/or luciferase reporter assays in immune cell types. Co-immunoprecipitation, ChIP-qPCR, and 3C-qPCR were performed on patient-derived EBV B cells homozygous for the non-risk or SLE risk TNFAIP3 haplotype to assess haplotype-specific effects on transcription factor binding and chromatin regulation at the TNFAIP3 locus. This study found that the TNFAIP3 locus has a complex chromatin regulatory network that spans â¼1M bp from the promoter region of IL20RA to the 3' untranslated region of TNFAIP3. Functional dissection of the enhancer demonstrated co-dependency of the RelA/p65 and CEBPB binding motifs that, together, increase IL20RA and IFNGR1 expression and decreased TNFAIP3 expression in the context of the TNFAIP3 SLE risk haplotype through dynamic long-range interactions up- and downstream. Examination of SNPs in linkage disequilibrium (D' = 1.0) with rs10499197 identified rs9494868 as a functional SNP with risk allele-specific increase in nuclear factor binding and enhancer activation in vitro. In summary, this study demonstrates that SNPs carried on the â¼109 kb SLE risk haplotype facilitate hypermorphic IL20RA and IFNGR1 expression, while suppressing TNFAIP3 expression, adding to the mechanistic potency of this critically important locus in autoimmune disease pathology.
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Sjögren's disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren's cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.
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Estudio de Asociación del Genoma Completo , Síndrome de Sjögren , Predisposición Genética a la Enfermedad , Humanos , Polimorfismo de Nucleótido Simple , Síndrome de Sjögren/genéticaRESUMEN
OBJECTIVE: Genetic variants spanning UBE2L3 are associated with increased expression of the UBE2L3-encoded E2 ubiquitin-conjugating enzyme H7 (UbcH7), which facilitates activation of proinflammatory NF-κB signaling and susceptibility to autoimmune diseases. We undertook this study to delineate how genetic variants carried on the UBE2L3/YDJC autoimmune risk haplotype function to drive hypermorphic UBE2L3 expression. METHODS: We used bioinformatic analyses, electrophoretic mobility shift assays, and luciferase reporter assays to identify and functionally characterize allele-specific effects of risk variants positioned in chromatin accessible regions of immune cells. Chromatin conformation capture with quantitative polymerase chain reaction (3C-qPCR), chromatin immunoprecipitation (ChIP)-qPCR, and small interfering RNA (siRNA) knockdown assays were performed on patient-derived Epstein-Barr virus-transformed B cells homozygous for the UBE2L3/YDJC nonrisk or risk haplotype to determine if the risk haplotype increases UBE2L3 expression by altering the regulatory chromatin architecture in the region. RESULTS: Of the 7 prioritized variants, 5 demonstrated allele-specific increases in nuclear protein binding affinity and regulatory activity. High-throughput sequencing of chromosome conformation capture coupled with ChIP (HiChIP) and 3C-qPCR uncovered a long-range interaction between the UBE2L3 promoter (rs140490, rs140491, rs11089620) and the downstream YDJC promoter (rs3747093) that was strengthened in the presence of the UBE2L3/YDJC risk haplotype, and correlated with the loss of CCCTC-binding factor (CTCF) and gain of YY1 binding at the risk alleles. Depleting YY1 by siRNA disrupted the long-range interaction between the 2 promoters and reduced UBE2L3 expression. CONCLUSION: The UBE2L3/YDJC autoimmune risk haplotype increases UBE2L3 expression through strengthening a YY1-mediated interaction between the UBE2L3 and YDJC promoters.
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Enfermedades Autoinmunes/genética , Factor de Unión a CCCTC/fisiología , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/fisiología , Factor de Transcripción YY1/fisiología , Regulación de la Expresión Génica , Variación Genética , Haplotipos , Humanos , Factores de RiesgoRESUMEN
OBJECTIVE: Genetic variants in the region of tumor necrosis factor-induced protein 3-interacting protein 1 (TNIP1) are associated with autoimmune disease and reduced TNIP1 gene expression. The aim of this study was to define the functional genetic mechanisms driving TNIP1 hypomorphic expression imparted by the systemic lupus erythematosus-associated TNIP1 H1 risk haplotype. METHODS: Dual luciferase expression and electrophoretic mobility shift assays were used to evaluate the allelic effects of 11 risk variants on enhancer function and nuclear protein binding in immune cell line models (Epstein-Barr virus [EBV]-transformed human B cells, Jurkat cells, and THP-1 cells), left in a resting state or stimulated with phorbol 12-myristate 13-acetate/ionomycin. HiChIP was used to define the regulatory 3-dimensional (3-D) chromatin network of the TNIP1 haplotype by detecting in situ long-range DNA contacts associated with H3K27ac-marked chromatin in EBV B cells. Then, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the expression of genes within the 3-D chromatin network. RESULTS: Bioinformatics analyses of 50 single-nucleotide polymorphisms on the TNIP1 H1 risk haplotype identified 11 non-protein-coding variants with a high likelihood of influencing TNIP1 gene expression. Eight variants in EBV B cells, 5 in THP-1 cells, and 2 in Jurkat cells exhibited various allelic effects on enhancer activation, resulting in a cumulative suppressive effect on TNIP1 expression (net effect of risk variants -7.14 fold, -6.80 fold, and -2.44 fold, respectively; n > 3). Specifically, in EBV B cells, only 2 variants (rs10057690 and rs13180950) exhibited allele-specific loss of both enhancer activity and nuclear protein binding (each P < 0.01 relative to nonrisk alleles). In contrast, the rs10036748 risk allele reduced binding affinities of the transcriptional repressors basic helix-loop-helix family member 40/differentially expressed in chondrocytes 1 (bHLHe40/DEC1) (P < 0.05 relative to nonrisk alleles) and CREB-1 (P not significant) in EBV B cells, resulting in a gain of enhancer activity (P < 0.05). HiChIP and qRT-PCR analyses revealed that overall transcriptional repression of the TNIP1 haplotype extended to the neighboring genes DCTN4 and GMA2, both of which also showed decreased expression in the presence of the TNIP1 risk haplotype (P < 0.001 and P < 0.01, respectively, relative to the nonrisk haplotype); notably, it was found that these genes share a 3-D chromatin network. CONCLUSION: Hypomorphic TNIP1 expression results from the combined concordant and opposing effects of multiple risk variants carried on the TNIP1 risk haplotype, with the strongest regulatory effect in B lymphoid lineage cells. Furthermore, the TNIP1 risk haplotype effect extends to neighboring genes within a shared chromatin network.
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Proteínas de Unión al ADN/genética , Lupus Eritematoso Sistémico/genética , Linfocitos B , Cromatina , Expresión Génica , Haplotipos , Humanos , Medición de RiesgoRESUMEN
Genome-wide association studies (GWAS) and fine mapping studies in autoimmune diseases have identified thousands of genetic variants, the majority of which are located in non-protein-coding enhancer regions. Enhancers function within the context of the three-dimensional (3D) genome to form long-range DNA looping events with target gene promoters that spatially and temporally regulate gene expression. Investigating the functional significance of GWAS variants in the context of the 3D genome is essential for mechanistic understanding of these variants and how they influence disease pathology by altering DNA looping between enhancers and the target gene promoters they regulate. In this review, we discuss the functional complexity of the 3D genome and the technological approaches used to characterize DNA looping events. We then highlight examples from the literature that illustrate how functional mapping of the 3D genome can assist in defining mechanisms that influence pathogenic gene expression. We conclude by highlighting future advances necessary to fully integrate 3D genome analyses into the functional workup of GWAS variants in the continuing effort to improve the health of patients with autoimmune diseases.
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Cromatina/química , Cromatina/genética , Predisposición Genética a la Enfermedad/genética , Genoma Humano/genética , Estudio de Asociación del Genoma Completo/métodos , Conformación Molecular , Humanos , Regiones Promotoras Genéticas/genéticaRESUMEN
Genetic variants can confer risk to complex genetic diseases by modulating gene expression through changes to the epigenome. To assess the degree to which genetic variants influence epigenome activity, we integrate epigenetic and genotypic data from lupus patient lymphoblastoid cell lines to identify variants that induce allelic imbalance in the magnitude of histone post-translational modifications, referred to herein as histone quantitative trait loci (hQTLs). We demonstrate that enhancer hQTLs are enriched on autoimmune disease risk haplotypes and disproportionately influence gene expression variability compared with non-hQTL variants in strong linkage disequilibrium. We show that the epigenome regulates HLA class II genes differently in individuals who carry HLA-DR3 or HLA-DR15 haplotypes, resulting in differential 3D chromatin conformation and gene expression. Finally, we identify significant expression QTL (eQTL) x hQTL interactions that reveal substructure within eQTL gene expression, suggesting potential implications for functional genomic studies that leverage eQTL data for subject selection and stratification.
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Cromatina/genética , Haplotipos/genética , Desequilibrio de Ligamiento/genética , Sitios de Carácter Cuantitativo/genética , Femenino , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , MasculinoRESUMEN
RATIONALE: We previously reported that vascular endothelial growth factor (VEGF)-induced binding of VEGF receptor 2 (VEGFR2) to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2. However, the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. OBJECTIVE: The goals for the present study were as follows: (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain whether such determinants were critical for physiological angiogenesis in vivo. METHODS AND RESULTS: Structural modeling uncovered 2 novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Furthermore, we found that the VEGF-induced VEGFR2-epsin interaction promoted casitas B-lineage lymphoma-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties in vitro, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing. CONCLUSIONS: Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Neovascularización Fisiológica/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Sistemas de Liberación de Medicamentos/tendencias , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Tumor angiogenesis is critical for cancer progression. In multiple murine models, endothelium-specific epsin deficiency abrogates tumor progression by shifting the balance of VEGFR2 signaling toward uncontrolled tumor angiogenesis, resulting in dysfunctional tumor vasculature. Here, we designed a tumor endothelium-targeting chimeric peptide (UPI) for the purpose of inhibiting endogenous tumor endothelial epsins by competitively binding activated VEGFR2. We determined that the UPI peptide specifically targets tumor endothelial VEGFR2 through an unconventional binding mechanism that is driven by unique residues present only in the epsin ubiquitin-interacting motif (UIM) and the VEGFR2 kinase domain. In murine models of neoangiogenesis, UPI peptide increased VEGF-driven angiogenesis and neovascularization but spared quiescent vascular beds. Further, in tumor-bearing mice, UPI peptide markedly impaired functional tumor angiogenesis, tumor growth, and metastasis, resulting in a notable increase in survival. Coadministration of UPI peptide with cytotoxic chemotherapeutics further sustained tumor inhibition. Equipped with localized tumor endothelium-specific targeting, our UPI peptide provides potential for an effective and alternative cancer therapy.
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Proteínas Adaptadoras del Transporte Vesicular/farmacología , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Péptidos/farmacología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencias de Aminoácidos , Animales , Ratones , Ratones Noqueados , Metástasis de la Neoplasia , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Péptidos/genética , Péptidos/metabolismo , Estructura Terciaria de Proteína , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Uncontrolled canonical Wnt signalling supports colon epithelial tumour expansion and malignant transformation. Understanding the regulatory mechanisms involved is crucial for elucidating the pathogenesis of and will provide new therapeutic targets for colon cancer. Epsins are ubiquitin-binding adaptor proteins upregulated in several human cancers; however, the involvement of epsins in colon cancer is unknown. Here we show that loss of intestinal epithelial epsins protects against colon cancer by significantly reducing the stability of the crucial Wnt signalling effector, dishevelled (Dvl2), and impairing Wnt signalling. Consistently, epsins and Dvl2 are correspondingly upregulated in colon cancer. Mechanistically, epsin binds Dvl2 via its epsin N-terminal homology domain and ubiquitin-interacting motifs and prohibits Dvl2 polyubiquitination and degradation. Our findings reveal an unconventional role for epsins in stabilizing Dvl2 and potentiating Wnt signalling in colon cancer cells to ensure robust colon cancer progression. The pro-carcinogenic role of Epsins suggests that they are potential therapeutic targets to combat colon cancer.
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Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Adenocarcinoma/genética , Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , Fosfoproteínas/genética , Vía de Señalización Wnt/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adenocarcinoma/inducido químicamente , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Azoximetano , Sitios de Unión , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis/patología , Colon/metabolismo , Colon/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteínas Dishevelled , Células HT29 , Humanos , Ratones , Ratones Noqueados , Fosfoproteínas/metabolismo , Cultivo Primario de Células , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Dodecil Sulfato de Sodio , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell-specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Sistema Linfático/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Alelos , Animales , Proliferación Celular , Separación Celular , Cruzamientos Genéticos , Endocitosis , Células Endoteliales/citología , Femenino , Citometría de Flujo , Proteínas Fluorescentes Verdes/química , Indoles/química , Ligandos , Ganglios Linfáticos/patología , Linfedema/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Naftalenos/química , Plásmidos/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.
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Endosomas/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Hiperinsulinismo/metabolismo , Resistencia a la Insulina , Insulina/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Animales , Endosomas/genética , Transportador de Glucosa de Tipo 4/genética , Humanos , Hiperinsulinismo/genética , Ratones , Transporte de Proteínas , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Proteínas de Unión al GTP rab5/genéticaRESUMEN
OBJECTIVE: We previously showed that endothelial epsin deficiency caused elevated vascular endothelial growth factor receptor 2 (VEGFR2) and enhanced VEGF signaling, resulting in aberrant tumor angiogenesis and reduced tumor growth in adult mice. However, direct evidence demonstrating that endothelial epsins regulate angiogenesis specifically through VEGFR2 downregulation is still lacking. In addition, whether the lack of epsins causes abnormal angiogenesis during embryonic development remains unclear. APPROACH AND RESULTS: A novel strain of endothelial epsin-deleted mice that are heterozygous for VEGFR2 (Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre mice) was created. Analysis of embryos at different developmental stages showed that deletion of epsins caused defective embryonic angiogenesis and retarded embryo development. In vitro angiogenesis assays using isolated primary endothelial cells (ECs) from Epn1(fl/fl); Epn2(-/-); iCDH5 Cre (EC-iDKO) and Epn1(fl/fl); Epn2(-/-); Flk(fl/+); iCDH5 Cre (EC-iDKO-Flk(fl/+)) mice demonstrated that VEGFR2 reduction in epsin-depleted cells was sufficient to restore normal VEGF signaling, EC proliferation, EC migration, and EC network formation. These findings were complemented by in vivo wound healing, inflammatory angiogenesis, and tumor angiogenesis assays in which reduction of VEGFR2 was sufficient to rescue abnormal angiogenesis in endothelial epsin-deleted mice. CONCLUSIONS: Our results provide the first genetic demonstration that epsins function specifically to downregulate VEGFR2 by mediating activated VEGFR2 internalization and degradation and that genetic reduction of VEGFR2 level protects against excessive angiogenesis caused by epsin loss. Our findings indicate that epsins may be a potential therapeutic target in conditions in which tightly regulated angiogenesis is crucial, such as in diabetic wound healing and tumors.